1. Field of the Invention
The invention relates to animal models for the growth and treatment of human neurally-derived tumors and the treatment of neurofibrosarcoma tumors using a therapeutic regimen which inhibits angiogenesis and tumor vascularization.
2. Description of the Background Art
Neurofibromatosis is an autosomal dominant genetic disorder associated with the development of multiple benign tumors and occasional malignant tumors. No effective treatment is available for either form of tumor and the malignant tumors, neurofibrosarcomas, are usually fatal despite aggressive surgical, medical, and radiotherapeutic treatment regimens (Martuza, R. L., Neurosurgery, MacGraw-Hill, Vol. 1, 1984, pp. 511-521). Therefore, new approaches for the treatment of neurofibrosarcomas would be of enormous benefit at this time.
It has become increasingly evident that angiogenesis, the formation of blood vasculature, is a fundamental and necessary event in the growth of solid tumors (Brem, S., CNS 23:440-453 (1976); Folkman, J. et al., J. Exp. Med. 133:275-288 (1971); Folkman, J., Ann. Surg. 175:409-416 (1972); Folkman, J., Adv. Cancer Res. 43:175-203 (1985); Folkman, J. et al., Science 235:442-447 (1987); Greenblatt, M. et al., J. Nat. Cancer Inst. 41:111-124 (1968); Klagsbrun, M. et al., Cancer Res. 36:110-114 (1976); Rastinejad, F. et al., Cell 56:345-355 (1989); Tannock, J. F., Br. J. Cancer 22:258-273 (1968); Tannock, J. F., Canc. Res. 30:2470-2476 (1970); Thompson, J. A. et al., Science 241:1349-1352 (1988). Zagzag, D. et al., Am. J. Pathol. 131:361-372 (1988); Ziche, M. et al., JNCI 69:483-487 (1982)). Thus, therapeutic strategies directed at disrupting this process are expected to be important. Following establishment of a blood supply, tumor cells not only begin to grow but also acquire the potential for metastasizing to distant sites by entering the circulation through this new vasculature (Folkman, J., Adv. Cancer Res., supra). Invasiveness of neoplastic cells in several in vitro, in vivo, and in situ models has indeed been linked to angiogenesis (Brem, S., Proc. Amer. Assoc. Neurol. Surgeons Ann. Meet. Washington, D.C., 1989, p.382 (abst)).
Tumor angiocyenesis is induced by soluble tumor angiogenesis factors produced by tumor cells (Folkman, J. et al., 1971, supra; Folkman, J., 1985, supra). Several angiogenic factors, such as the fibroblast growth factors (xcex1FGF and xcex2FGF), angiogenin, and the transforming growth factors, TGF-xcex1 and TGF-xcex2, have been purified, their amino acid sequences detennined, and their genes cloned (Folkman, J. et al., 1987, supra). These studies led to a hypothesis that solid tumors are angiogenesis-dependent, and that xe2x80x9canti-angiogenesisxe2x80x9d was a potential approach to tumor therapy.
The art of angiogenesis research has relied mainly on three models for the ini vivo study of capillary proliferation: (1) The rabbit and rodent cornea micropocket; (2) the chicken embryo chorloallantoic membrane; and (3) the hamster cheek pouch for murine experimental tumors (Folkman, J., 1985, supra; Folkman, J. et al., 1987, supra; Greenblatt, M. et al., supra; and Zagzag, D. et al., supra).
In 1983, Folkman""s group disclosed that heparin or a heparin fragment administered with cortisone, caused regression of large tumor masses and prevented metastases (Folkman, J. et al., Science 221:719-725 (1983)). Angiogenesis was inhibited when heparin, or one of its fragments lacking anti-coagyulant activity, was administered simultaneously with an angiostatic steroid. This was somewhat paradoxical given the fact that heparin alone actually promotes angiogenesis in vivo and can potentiate endothelial locomotion and proliferation in vitro. The angiostatic steroids by themselves had weak or no angiogenesis-inhibiting activity (Crum, R. et al., Science 230:1375-1378 (1985)). Potent inhibition of angiogenesis required the xe2x80x9cpairxe2x80x9d effect of two components.
Despite the promise of this approach, the literature reflects disparate results; some investigators have observed inhibition oftumor growth with heparin plus cortisone whereas others have not (Lee, K. et al., Canc. Res. 47:5201-5204 (1987); Penhaglion, M. et al., . JNCI 74:869-873 (1985); Rorg, G. H. et al., Cancer 57:586-590 (1986); Sakamoto, N. et al., JNCI 78:581-585 (1987); and Ziche, M. et al., Int. J. Cancer 35:549-552 (1985)).
For tumors that were responsive to heparin and cortisone, oral administration of 200 units of heparin per ml of drinking water was generally found to be the minimum effective dose, and tumor regression was more rapid as the dose increased up to 1000 units/ml. However, when the heparin dose was increased further, for example, to 2000-5000 units/ml, rapid tumor growth rather than regression was observed (Crum, R. et al., 1985, supra).
The efficacy of heparin was found to depend critically on the source of the heparin. The most potent, PanheparinR (Abbott Laboratories), is no longer commercially available. The next most potent heparin, from Hepar, Inc., Franklin, Ohio) was noted to cause regression of reticulum cell sarcoma, but not of Lewis lung carcinoma, in mice (Folkman, J. et al., 1983, supra). Heparin preparations are frequently heterogeneous in composition, molecular size, sequence, and position of substituents (N-sulfate, O-sulfate, and glucuronic acid). This may account for the differences in anti-tumor efficacy when the heparin is used in combination with angiostatic steroids (Folkman, J. et al., Science 243:1490-1493 (1989)). However, these reports tested only malignant murine tumors which may be particularly resistant to such therapy. The ability to test anti-angiogenic agents, alone or in combination, on human tumors would be of great benefit for devising therapeutic strategies.
Animal models are important tools for studying the growth and spread of human tumors and for developing and testing therapeutic strategies. The development of congenitally athymic xe2x80x9cnudexe2x80x9d mice and refinement of techniques for producing immunodeficiencies in rodents have permitted more detailed study of a variety of xenotransplanted human tumors (Aamdal, S. et al., Int. J. Cancer 34:725-730 (1984); Abernathey, C. D. et al., Neurosiirgery 22:877-881 (1988); Bailey, M. J. et al., Br. J. Cancer 50:721-724 (1984); Bigner, S. H. et al., .J. Neuropathol. Exp. Neurol. 40:390-409 (1981); Dumont, P. et al., Int. J. Cancer 33:447-451 (1984); Epstein, A. L. et al., Cancer 37:2158-2176 (1976); Giovanella, B. et al., Adv. Cancer Res. 44:69-129 (1985); Rajnay, J. et al., Oncology 44:307-311 (1987); Rao, M. S. et al., .J Pathology 135:169-177 (1981); Schold, S. C. et al., Prog. Exp. Tumor Res. 28:18-31 (1984)).
Several human brain tumor models involving subcutaneous (s.c.) or intracerebral (i.c.) implants in nude mice, have been reported (Basler, G. A. et al., in The Nude Mouse in Experimental and Clinical Research, Fogh, J. et al. (eds.), New York: Academic Press, Vol. 2, pp. 475-490 (1982); Bradley, N. J. et al., Br. J. Cancer 38:263-272 (1978); Bullard, D. E. et al., Neurosurgery 4:308-314 (1979); Horten, B. C. et al., J. Neuropathol. Exp. Neurol. 40:493-511 (1981); O""Sullivan, J. P. et al., J. Endocr. 79:139-140 (1978); Rana, M. W. et al., Proc. Soc. Exp. Biol. Med. 155:85-88(1977); Shapiro, W. R. et al., J. Natl. Cancer Inst . 62:447-453 (1979); Slagel, D. E. et al., Cancer Res. 42:812-816 (1982); Tueni, E. A. et al., Eur. J. Cancer Clin. Oncol. 28:1163-1167 (1987); Ueyama, Y et al., Br. J. Cancer 37:644-647 (1978)).
In vivo models oftumors grown in nude mice have been extremely useful for a wide variety of purposes, such as studying tumor biology and testing, sensitivity to chemotherapy and radiotherapy. Although s.c. xenografts allow serial tumor volume measurements, and the procedureof implantation is easy, the s.c. tumors are difficult to measure precisely with calipers since they may be surrounded by fibrous tissue or fat. Many neural tumors typically grow slowly. A short-term method for the growth of particular human tumors in mice was developed by Castro and Cass (Castro, J. E. et al., Br. J. Surg. 61:421-426 (1974)) and later refined by Bogden et al. (Bodgen, A. E. et al., Cancer 48:10-20 (1981)), using solid tumor fragments implanted under the kidney capsule.
The sub-renal capsule assay allows precise measurement (accurate to 0.1 mm) of changes in tumor size as measured using a stereomicroscope with an ocular micrometer. The sub-renal capsule contains no fat and minimal, if any, reactive fibrous tissue. Greater blood flow has been observed in sub-renal capsule tumor implants as compared to s.c. tumors, suggesting an advantage of the sub-renal capsule for delivery of both nutrients and systemically administered antitumor agents (Sands, H. et al., Cancer Lett. 24:65-72 (1984)). In addition, invasive and metastatic properties, not generally observed after s.c. implantation, may be observed with tumors growing under the renal capsule.
Intracerebral tumormodels arethoughtto mimic the clinical environment for neurally-derived tumors. However, the presence of the blood-brain barrier makes drug delivery and limitations thereto an important concern. Furthermore, studies are usually limited to only one point, survival. In contrast, sub-renal capsule tumors are accessible for direct, accurate and repeated measurements. More importantly, the extracerebral location allows investigation of the cellular sensitivity of these neoplasms to drugs with less concern about the drug delivery problem. If a tumor cell is inherently resistant to an agent, improving delivery of that agent into the brain will be of no benefit. Demonstration of cellular sensitivity of a sub-renal capsule tumor to an agent can be translated into an approach for intracerebral delivery of that agent (Schold, S. C. et al., Prog. Exp. Tumor Res. 28:18-31 (1984)).
Meningiomas are important and interesting examples of tumorigenesis in the human nervous system because: (1) they arerelatively common and clinically important; (2) a 3:1 female:male incidence ratio suggests possible hormonal modulation; and (3) recent molecular genetic studies have demonstrated an associated abnormality on chromosome 22 (Jay, J. R. et al., J. Neurosurg. 62:757-762 (1985); Kaplan, J. C. et al., J. Med. Genet. 24:65-78 (1987); Magdelenat, H. et al., Acta Neurochir. 64:199-213 (1982); Markwalder, M. T. et al., Surg. Neurol. 30:97-101 (1988); Martuza, R. L. et al., Neurosurgery 9:665-671 (1981); Mirimanoff, R. O. et al., J. Neurosurg. 62:18-24 (1985); Olson, J. J. et al., J. Neurosurg. 66:584-587 (1987); Olson, J. J. et al., J. Neurosurg. 65:99-107 (1986)).
Another neurally-derived tumor, the Schwannoma, appears most frequently in humans as an acoustic neuroma which is a benign tumor formed by Schwann cells of the eighth cranial nerve (Rubinstein, L. J., Armed Forces Institute of Pathology, Washington, D.C., pp. 205-214 (1972)). Most acoustic neuromas occur unilaterally in a sporadic, non-hereditary fashion, whereas bilateral acoustic neuromas are associated with neurofibromatosis-2 (NF2), a serious autosomal dominant genetic disorder associated with multiple Schwannomas, neurofibromas, acoustic neuromas, ependymomas, and astrocytomas (Martuza, R. L. et al., N. Engl. J. Med. 318:684-688 (1988)). Recent molecular genetic studies have revealed that both unilateral nonhereditary acoustic neuromas as well as those associated with NF2 are related to a deletional mutation or an inactivating mutation of a putative growth suppressor gene on chromosome 22 (Seizinger, B. R. et al., Nature 322:644-647 (1986); Seizinger, B. R. et al., Science 236:317-319 (1987)). This gene has not yet been cloned, and its function is unknown. However, it would be important to have a model system for study and genetic manipulation of the growth of human acoustic neuromas. To date, no such model exists.
Clinically, acoustic neuromas are the most common tumors in the cerebello-pontine angle, and despite modern surgical and anesthetic techniques, they remain a serious surgical challenge. Although most can be totally removed, some are very vascular or recurrent. This is particularly problematic in the NF2 patient who faces the possibility of bilateral deafness, facial paresis, or other neurologic dysfunction. No medical treatment is currently of proven efficacy to modify the growth of these tumors (Martuza, R. L. et al., Neurosurgery 10:1-12 (1982); Martuza, R. L. et al., N. Engl. J. Med. 318:684-688 (1988)).
The study of Schwannomas is also important from the standpoint of Schwann cell biology and molecular mechanisms of growth control. Two endogenous mitogens have been demonstrated to stimulate Schwann cell mnitosis. One is a membrane-associated mitogen found in the neurite axon and the other, termed glial growth factor (GGF), is a soluble mitogen normally found in the pituitary and in the caudate nucleus. Human acoustic neuromas have very high levels of a mitogen similar to, or identical with, GGF, and suggest the possibility that the growth of Schwannomas is self-stimulated by an autocrine mechanism (Martuza, R. L. et al., TINS 11:22-27 (1988); Martuza, R. L. et al., N. Engl. J. Med. 318:684-688 (1988)). Other studies have demonstrated elevated levels of FGF mRNA in acoustic neuromas (Murphy, P. R. et al., Mol. Endocrinol. 3:225-231 (1989)).
Finally, the study of Schwannomas is important for our understanding of the genetic mechanisms oftumorigenesis. Studies both of NF2 bilateral acoustic neuromas as well as of non-hereditary unilateral acoustic tumors suggest that both types of acoustic neuromas are related to a loss or inactivation of a tumor suppressor gene on the long arm of chromosome 22 (Seizinger, B. R. et al., 1986, 1987, supra).
To date, there are no useful techniques for studying the growth behavior of Schwannomas in a controlled laboratory setting. Study of human brain tumors in vitro has limitations. For example, studies based on monolayer cell culture or organ culture do not reflect in vivo morphology, neovascularization, or growth characteristics of the original tumor (Horten, B. C. et al., J. Neuropathol. Exp. Neurol. 40:493-511 (1981)). Appenzeller et al. (Appenzeller, O. et al., J. Neurol. Sci. 74:69-77 (1986)) transplanted Schwann cells of the aural nerve, neurofibrosarcomas, and amalignant Schwannoma from three neurofibromatosis patients into sciatic nerves of immunosuppressed mice. However, that study was designed to evaluate myelination, not tumor formation.
In summary, there is an acute need in the art for animal models of human neurally-derived tumors for basic understanding of tumor growth regulation, tumor metastasis, as well as for development of treatment modalities. Furthermore, novel forms of therapy of human neurally-derived tumors such as neurofibrosarcomas are urgently needed.
The present invention is based on the inventors"" recognition that malignant tumors of peripheral nerve are associated with neo-vascularity, and that therapeutic strategies targeted to tumor angiogenesis should be efficacious in treating such tumors which are associated with neurofibromatosis in humans.
In one embodiment, the invention is directed to a nude mouse model system to evaluate the effects of therapeutics on human neurally-derived tumors. This model comprises growth oftumor cells derived from the fresh tumor or from cultured tumor cells under the renal capsule of the nude mouse. The invention is directed to the use of this animal model to evaluate therapeutic agents. In a preferred embodiment, the therapeutic agent is a combination of an angiostatic steroid and heparin or a fragment or synthetic substitute thereof. By inhibiting angiogenesis and neovascularization, this agent inhibits tumor growth.
The invention is directed in particular to a method for treatingy a neurofibrosarcoma comprising the administration of an effective amount of heparin, a heparin fragment, or a synthetic heparin substitute and an anLriostatic steroid or steroid derivative to an animal in need of such treatment.